The present invention concerns methods of screening for active agents useful as an antibacterial agent against Pseudomonas, as well as active agents and methods of use thereof in treating Pseudomonas. 
Pseudomonas infection is a leading cause of death in cystic fibrosis, and one of the top causes of serious hospital-acquired infections. Pseudomonas infection is particularly serious in burn victims and leukemia patients, and can cause blindness by infection in patients afflicted with trauma to the eye through surgery or contact lenses. Further, Pseudomonas has a high intrinsic resistance to current antibiotics. Hence, there is a need for the development of new antibiotics to treat Pseudomonas infections.
The use of antisense oligonucleotides in the treatment of bacterial infections is known. U.S. Pat. No. 5,294,533 to J. Lupski and L. Katz (assigned to Baylor College of Medicine and Abbott Laboratories, Inc.) describes a method of interrupting the expression of a macromolecular synthesis operon in bacteria comprising the step of binding an antisense oligonucleotide to a single stranded DNA or to a mRNA transcribed from the macromolecular synthesis operon. The antisense oligonucleotide can be either sequence specific to a unique intergenic sequence or a sequence specific to a bacterial homologous sequence. By interrupting the expression of the macromolecular synthesis, it is said that bacterial infections can be treated.
U.S. Pat. No. 6,060,241 to I. Corthesy-Theulaz (assigned to Kieta Holding SA) describes a poly-3-hydroxybutyrate metabolic pathway essential for Helicobacter pylori survival in a host. A Helicobacter pylori Coenzyme A transferase (Hp CoA-t), thiolase and PHB synthase, as well as methods for their preparation and use are provided. Pharmaceutical compositions containing Hp CoA-t protein fragments, antisense nucleic acids or other inhibitors of Hp CoA-t, thiolase and PHB synthase, as well as methods for their use in the treatment of some types of gastric disease are also described. This reference is not concerned with Pseudomonas. 
PCT Application WO98/03533A1 to A. Arrow et al. (assigned to Oligos Etc. and Oligos Therapeutics Inc.) describes the general therapeutic use of nuclease resistant oligonucleotides for treating animals having an infection caused by a pathogenic bacterium. The method is a general one and involves the integration of (1) methods for selecting the correct oligonucleotide, (2) synthesis and purification of nuclease resistant oligonucleotides, and (3) methods for in vitro analysis of potential antimicrobial oligonucleotides.
There remains a need for new ways to screen for antibiotics effective against Pseudomonas, along with compounds and methods of treating Pseudomonas infections.
The present invention provides a method of screening for compounds that inhibit the virulence of Pseudomonas bacteria. The method comprises the steps of: providing a culture medium comprising Pseudomonas bacteria; administering a test compound to said bacteria; and then detecting the presence or absence of inhibition of the catabolite repression control (Crc) protein in said bacteria, the inhibition of the Crc protein indicating said compound has antivirulence activity against Pseudomonas bacteria.
A second aspect of the present invention is an antivirulence compound (for example, an antisense oligonucleotide) that inhibits expression or activity of the Crc protein in a Pseudomonas bacteria. Such compounds are useful as antivirulence compounds. When such compounds are antisense oligonucleotides they are preferably from 8 to 25, 40 or 80 nucleotides in length, and preferably are nuclease resistant.
A third aspect of the present invention is an antivirulence compound as described above in a pharmaceutically acceptable carrier.
A fourth further aspect of the present invention is a method of inhibiting the virulence of Pseudomonas bacteria, comprising administering to Pseudomonas bacteria an antivirulence compound as described above in an effective antivirulence amount. The administering step may be carried out in vitro, for example in drug testing or screening studies, or may be carried out in vivo in the treatment of a subject.
A further aspect of the present invention is a method of treating Pseudomonas infection in a subject in need thereof, comprising administering to said subject an antivirulence compound as described above in an amount effective to treat said Pseudomonas infection.
A still further aspect of the present invention is the use of an antivirulence compound as described above for the preparation of a medicament for carrying out a method as described above.
The foregoing and other objects and aspects of the present invention are explained in greater detail in the specification set forth below.